This application describes a research program to characterize the process of transcription by E. Coli RNA polymerase molecules at the single molecule level by means of novel biophysical methods of optical trapping and nanometer-level position sensing. The ultimate objective of this proposal is to investigate the mechanochemical properties of this processive enzyme, to gain an understanding of how the DNA-direct RNA synthesis is coupled to translocation along the DNA template. The specific aims of this proposal are: 1) To determine the force- velocity relationship for RNAP and characterize themechanochemical properties of this motor protein. These studies will investigate the mechanochemical behavior of this enzyme spanning the full range of external load forces; i.e., below, at and above the stall force of the motor. Similarly, the force- velocity relationship of the enzyme working in the presence of an external load applied in the direction of the motion will be characterized. 2) Investigate transcriptional pausing, stalling and arrest by RNAP and its relationship to the nature of the DNA template. These experiments will investigate whether the arrest of the enzyme is a probabilistic or a deterministic phenomenon, whether it is dictated by the sequence of the template, and if reversible and irreversible arrests are influenced by the present and the magnitude of the external load. The sequence effects onpausing and stalling will be studied on complex sequences, on direct repeat templates, and on homopolymers. Attempts will be also made to observe, directly, slippage under the external load. Finally, these studies will investigate what is the effect of transcript cleavage factors such as GreA and GreB on pausing and stalling. 3) Study the fluctuation behavior of the dynamics of individual transcribing enzymes. A variance analysis will be applied to obtain information about the number and duration of the rate-limiging steps in the enzyme reaction, as a way to get an insight into the molecular mechanism responsible for the mechanochemical coupling in RNAP. 4) Studies of the fine structure of the translocation of transcribing single RNAP molecules. These studies will be designed to I) investigate the effect of torsional strain during transcription, ii) obtain direct evidence of discontinuous or "inch worming" movement of the polymerase along the template, particularly during the early phases of transcription, and iii) attempt to resolve the process of transcription to single base pair resolution.